Project Summary Vector-borne diseases account for more than 17% of all infectious disease and cause more than 700,000 deaths annually1. Mosquitoes alone cause 400,000 malaria deaths and transmit viruses to hundreds of millions2. The vectorial capacity of mosquitoes depends on their ability to survive infection. The damaging effects of pathogenic invasion of the mosquito midgut are well-documented15-22, but little is known about how mosquitoes tolerate this stress. Intestinal stem cell (ISC) mediated midgut epithelial repair is essential for Drosophila survival following oral ingestion of pathogens33. The mosquito midgut epithelium contains ISC-like cells27, 34, 37-39, but their functional significance for infection outcomes and mosquito survival is unknown. We propose to address this knowledge gap in vector biology by investigating the mosquito gut regenerative response to pathogenic invasion. The ?black box? regarding the functional significance of ISCs in the mosquito midgut is part of a fundamental knowledge gap: physiological studies treat the mosquito midgut as a homogeneous whole, rather than a complex, regionally compartmentalized tissue comprised of multiple cell populations (e.g. enterocytes, enteroendocrine cells, and ISCs). The specific contributions of these cell types to gut-pathogen interactions have not been investigated. The proposed work will not only illuminate mosquito epithelial responses to infection at the cellular level but will lead to the creation of new and innovative tools for the broader vector biology community. The first two aims of this project are (A) to characterize gut epithelial cell dynamics in mosquitoes under conditions of homeostasis and oral infection and (B) to evaluate the role of midgut epithelial repair in mosquito infection outcomes. Aedes aegypti will be used as a model to determine what stimuli (including human pathogens) affect gut epithelial turnover rates, whether post-infection repair rebuilds the gut homeostatically or alters epithelial composition, what genetic pathways control midgut epithelial repair, and what role epithelial repair plays in vector survival and competence. Our third aim is (C) to determine the specific contributions of functionally differentiated cell populations to epithelial dynamics and infection response. We will use single-cell RNAseq/ATACseq to establish how many cell types compose the midgut epithelium, create new transgenic lines expressing fluorescent markers for important cell types (enteroendocrine cells and ISCs), and, using these lines, couple fluorescence-activated cell sorting with RNAseq to examine the transcriptional response of the three major cell types (enterocytes, enteroendocrine cells, and ISCs) to infection. Our study will fill a critical gap in our understanding of mosquito midgut regenerative responses to pathogenic invasion. Our long-term goal is to identify new targets for vector control strategies that disrupt gut regeneration and reduce survival of infected mosquitoes below the critical incubation threshold required for pathogen transmission. In addition to laying the groundwork for innovative control targets, we will create tools for the broader vector biology community, paving the way for novel discoveries in mosquito midgut physiology.